DTIC ADA434134: Synthesis of Lipoprotein Immunostimulants for Treating Prostate Cancer

0.71 MB·English

by  Defense Technical Information Center

AD
Award Number: DAMD17-02-1-0150
TITLE: Synthesis of Lipoprotein Immunostimulants for Treating
Prostate Cancer
PRINCIPAL INVESTIGATOR: Jerald C. Hinshaw, Ph.D.
CONTRACTING ORGANIZATION: Utah University
Salt Lake City, Utah 84102
REPORT DATE: January 2005
TYPE OF REPORT: Annual
PREPARED FOR: U.S. Army Medical Research and Materiel Command
Fort Detrick, Maryland 21702-5012
DISTRIBUTION STATEMENT: Approved for Public Release;
Distribution Unlimited
The views, opinions and/or findings contained in this report are
those of the author(s) and should not be construed as an official
Department of the Army position, policy or decision unless so
designated by other documentation.
20050603 252
O Form Approved
REPORT DOCUMENTATION PAGE 0OMB No. 074-0188
Public reporting burden for this collection of information is estimated to average 1 hour per response, Induding the time for reviewing instructions, searching existing data sources, gathering and maintaining
the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for
reducing this burden to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302, and to the Office of
Management and Budget, Paperwork Reduction Project (0704-0188), Washington, DC 20503
1. AGENCY USE ONLY 2. REPORT DATE 3. REPORT TYPE AND DATES COVERED
(Leave blank) January 2005 Annual (1 Jan 2004 -31 Dec 2004)
4. TITLE AND SUBTITLE 5. FUNDING NUMBERS
Synthesis of Lipoprotein Immunostimulants for Treating DAMD17-02-1-0150
Prostate Cancer
6. AUTHOR(S)
Jerald C. Hinshaw, Ph.D.
7. PERFORMING ORGANIZA TION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION
Utah University REPORT NUMBER
Salt Lake City, Utah 84102
E-Mail: Jerald. hinshaw@hsc .utah. edu
9. SPONSORING IMONITORING 10. SPONSORING / MONITORING
AGENCY NAME(S) AND ADDRESS(ES) AGENCY REPORT NUMBER
U.S. Army Medical Research and Materiel Command
Fort Detrick, Maryland 21702-5012
11. SUPPLEMENTARY NOTES
12a. DISTRIBUTION/AVAILABILITY STATEMENT 12b. DISTRIBUTION CODE
Approved for Public Release; Distribution Unlimited
13. ABSTRACT (Maximum 200 Words)
The prospects of stimulating a patient's own immune system as a therapeutic approach to'
the treatment of cancer in general, and prostate cancer in particular, is intriguing.
However, thus far immunotherapeutic approaches to the treatment of cancer (including
prostate cancer) in the clinical setting have not been uniformly successful. We are
chemically synthesizing molecular conjugates that comprise a Toll-Like Receptor (TLR)
ligand covalently linked to a prostate cancer tumor-associated antigen protein or peptide.
Using this tactic, we anticipate that the TLR-ligand portion of the conjugates will
stimulate dendritic cells (DCs) (as well as other TLR-expressing antigen presenting cells)
to secrete immune-activating cytokines. Concomitantly the tumor antigen component of the
complex will be processed and presented to activated T cells. In this way, a new and
potent immune system stimulation and antigen presentation mechanism aimed at the
stimulation of combined CD8+ and CD4+ T-cell responses, along with B-cell activation via
the innate/adaptive immune response connection, is being developed. We have prepared TLR-4
and TLR-7 ligands and are preparing mouse immunization experiments to test the efficacy of
this new approach to cancer immunotherapy.
14. SUBJECT TERMS 15. NUMBER OF PAGES
Prostate cancer immunotherapy, toll-like receptors, chemical 16
synthesis 16. PRICE CODE
17. SECURITY CLASSIFICATION 18. SECURITY CLASSIFICATION 19. SECURITY CLASSIFICATION 20. LIMITATION OF ABSTRACT
OF REPORT OF THIS PAGE OFABSTRACT
Unclassified Unclassified Unclassified Unlimited
NSN 7540-01-280-5500 Standard Form 298 (Rev. 2-89)
Prescribed by ANSI Std. Z39-18
298-102
Table of Contents
Cover ................................................ I
SF 298 ....................................... ............................................... 2
Table of Contents ............................................................................ 3
Introduction .................................................................................. 4
Body ............. ...I. .......................... 4
Key Research Accomplishments ........................................................ 10
Reportable Outcomes ...................................................................... 10
Conclusions ............................................ 10
References ...................................................................................... 11
Appendices ...................................................................................... 12
A. Introduction
In this project, we are developing a new technique for stimulating a patient's own immune
system as a therapeutic approach for the treatment of cancer in general, and prostate cancer,
in particular. Thus far, immunotherapeutic protocols for treating cancer (including prostate
cancer) in the clinical setting have not been uniformly successful1. Many of these methods
have been directed toward the stimulation of a CD8+ T-cell response in the host, but have
lacked a mechanism for vigorous immune system stimulation and have not combined
CD4+ T-cell responses or B-cell involvement2. Recently, techniques involving stimulation
of dendritic cells (DC) with tumor antigens have begun to show promise, but the process
involves isolation of DCs and in vitro stimulation, followed by re-injection into the patient.
Our methodology uses direct in vivo stimulation/maturation of DCs via Toll-like receptors
(TLRs)35. We are chemically synthesizing molecular conjugates that comprise a TLR
ligand covalently linked to a prostate cancer tumor-associated antigen protein or peptide.
Using this tactic, we anticipate that the TLR-ligand portion of the conjugates will stimulate
DCs (as well as other TLR-expressing antigen presenting cells) to secrete immune-
activating cytokines while the tumor antigen component of the complex will be processed
and presented to activated T cells. We have addressed the synthesis of conjugates based on
TLR-2 and TLR-7 ligands. The prostate cancer antigen portion of the complexes currently
includes an MHC Class I peptide epitope from prostate-specific membrane antigen
(PSMA)6 and a general MHC Class II peptide epitope (PADRE)7'8. We have included the
complete prostate-specific antigen (PSA) protein linked to a TLR-7 ligand. Our conjugates
represent a new and potent immune system stimulation and antigen presentation
mechanism with in vivo activation of DCs aimed at the stimulation of combined CD8+ and
CD4+ T-cell responses along with B-cell activation through the innate/adaptive immune
response connection. The conjugates are designed to elicit a vigorous immune response to
prostate cancer, and may be administered by s.c. injection, or possibly, nasal or oral routes.
B. Body
This section describes research accomplishments associated with the tasks outlined in the
original award application for Year Three of the project.
Task 1. Synthesize lipopeptide carriers covalently conjugated to PSA protein (months
1-14)
From previous effort we have completed the synthesis of the antigen conjugates in Figure 1
using the synthetic route outlined in Scheme 1.
4

in Scheme 2.
'N~ -/- 'S roeN
BrmactNm eNTHL0 7I~U~gfdj( TCLo(cid:127)NnR"7N-Ugaigteand
"HH
Molecular weight: = 28,700
Scheme 2. Preparation of the PSA Protein Conjugate
The PSMA immunostimulatory conjugates are based upon the human PSMA HLA-A2
restricted peptide epitope (ALFDIESKV) and incorporate an aminohexyl spacer between
the peptide epitope and a terminal cysteine for attachment to the TLR ligands. The PADRE
conjugates comprise the general CD4+ restricted peptide epitope PADRE7'8 using the
identical aminohexyl functionality bonded to a terminal cysteine (aK-clohexylalanine-
VAAWTLKAAa-aminohexyl-C). We also synthesized the PSMA epitope covalently
linked to the PADRE peptide through a central cysteine (aK-cyclohexylalanine-
VAAWTLKAAa-C-ALFDIESKV) in order to evaluate the immunogenicity of conjugates,
which contain both MHC Class I and MHC Class II epitopes. The TLR-2 ligand (PAM -
cys) lipopeptide conjugates proved to be quite insoluble, and thus far we have been unabl3e
to either adequately purify the materials or obtain mass spectral characterization data using
MALDI, ESI or FAB ionization techniques. Similar difficulties have been noted recently
for other PAM3-cys-peptide conjugates . For this reason, we are using the TLR-7-1igand
conjugates of Figure 1 and Scheme 2 in our immunization experiments.
Taskl2. Immunize HLA-DR4 and HLA-A2 transgenic mice with lipopeptide-PSA
conjugates. Analyze immune responses and test responses against prostate cancer
cell-lines (months 9-25)
Having prepared immunostimulating conjugates, we began mouse immunization
experiments. Founder homozygous mice carrying the HLA-A2.1 Enge transgene that
express the human class MHC Class I antigen HLA-A2. 1 on cells from the spleen, bone
marrow and thymus were obtained from Jackson laboratories, Bar Harbor, ME (Stock
Number: 003475). This surface protein recognizes the PSMA peptide epitope that is
incorporated into our conjugates. A breeding colony of these mice has been established at
conmjgmatsu. e nraelpzeo nes nd estresonse agins prstae cnce
The University of Utah. Since wc have included the generalized human and mouse MiC
Class II antigenic peptide epitope PADRE in our conjugate evaluation, it was not
necessary to utilize transgenic mice expressing human HLA-DR4 as originally proposed.
The PADRE peptide is expected to provide the desired CD4+ helper T-cell stimulation.
Mice in groups of three were immunized (s.c., axilla) with the TLR-7 ligand PSMA-
PADRE conjugate (Figure 2). Three levels of conjugate were initially examined (0.1, 1.0,
and 10 [tg). Mice were also immunized with the unconjugated PSMA, PADRE, and
PSMA-PADRE peptides. Syngenetic C57BL/6J mice that do not express the HLA-A2
gene were used as controls. Twenty-eight days after the initial immunization, each group
of mice received a boost vaccination. Eleven days post-boost, splenocytes and peripheral
lymph node (PLN) lymphocytes were harvested and [3H]-thymidine cell proliferation
assays were performed. Each cell population was administered [3H]-thymidine,
stimulated with antigenic peptides (10, 15, 7.5, and 3.8 jtg/ml) and controls, and incubated
for 24 h. Radioactivity incorporation compared to controls was taken as an indication of
the expansion of the cell population in response to antigenic peptide stimulation. Figure 3
summarizes our most recent results. Our previous work used a minimum of 0.1 jtg
vaccination. We therefore repeated the immunization protocol using a vaccination dose of
only 0.01 ltg. Figure 2 summarizes these results. It is apparent that even this low amount
of antigen also primes lymphocytes in this mouse model. This compares with Figure 4
which shows that even at 1000 times the amount (10 jtg) of the PSMA-PADRE peptide
(without the covalently attached TLR-7 ligand) elicits no response. This indicates the
powerful immunostimulatory effects of the Toll-like receptor-antigen conjugate approach
to cancer immunotherapy.
PLN Lymphocyte Proliferation Assay
24 hours
(0.01 gig TLR-7-PSMA-PADRE Vaccination)
-2500M
.0
E 2000 j ---- TLR-7
a -4-PSMA-PADRE
1500 PADRE
COI.L PSMA
1000 ---- Nonsense Peptide
500,- - Medium
E
0
0 10 20 30
Stimulant Concentration
(jig/ml)
Figure 2. 0.01 ýtg vaccination. Immune Response
7
PLN Lymphocyte Proliferation Assay
(10 microgram PSMA-PADRE Vaccination)
1000
o 900
800
C0L 76 00
SILW 6 _x_1 1\ - --PSMA-PADRE
M UX 500 Ai--P ADRE
0- 400 --X-PSMA
S300
-Medium
E 200
." 100
0
0 10 20 30 40
Peptide Stimulant Concentration
(microgram/ml)
Figure 3. 10 pýg PSMA-PADRE Peptide Immunization. No Immune Response
Interestingly, our most recent results with 0.1 ýtg immunization (Figure 4) show a clear
indication of the induction of tolerance to the powerful immune conjugate.
PLN Lymphocyte Proliferation Assay
24 hours
(0.1 gg TLR-7-PSMA-PADRE Vaccination)
3500
3000 - -e-TLR-7
a o 2500 - PSMA-PADRE
j
r 2000
-U--PADRE
"0.= - PSMA
E Eu 1500
1 000 Nonsense Peptide
S1000 - Medium
500
0 10 20 30
Stimulant Concentration
(G±g/ml)
Figure 4. 0.1 ýig Immunization. Induction of Tolerance
Figure 5 shows a non-transgenic mouse immunization control.
PLN Lymphocyte Proliferation Assay
24 hours
(Control Mice)
2500 _
C 2000 --- TLR-7
:0-P - PSMA-PADRE
•(cid:127) j 15o00
' Lo%. ,.J 0 0"0 ---PA R
CL-* PSMA
0.U -X-- Nonsense Peptide
C 500 -Medium
0 10 20 30
Stimulant Concentration
(Pig/ml)
Figure 5. Non-Transgenic Mouse Control. No Immune Response.
Preliminary chromium release experiments evaluating cytotoxic T lymphocyte killing of
LNCaP prostate cancer cells (which express the PSMA protein as well as being HLA-A2.1
positive) in culture did not work well at all (Figure 6) in that the response does not
smoothly follow the effector to target ratio.
Cell Lysis by
Vaccination-Induced CTLs
* 25
" 20
5 10
MCF-7
W 0 .
3.1:1 25.0:1 100.0:1
Effector to Target Ratio
Figure 6. Cell Lysis Experiment. LNCaP Lysis Data Are Not Reliable.
In preparation for these experiments, we experienced difficulty with the uptake and
retention of 51Cr by the LNCaP cells using standard labeling protocols. Our first approach
to the problem was to obtain different batches of the LNCaP (ATCC) cells. These cells
were also found to be difficult to 51Cr label. We then searched for an alternate human
prostate cancer cell line that expressed both PSA and PSMA as well as being HLA-A2.1
positive. Several cell lines were evaluated by immunostaining for the necessary markers
but we were unable to identify a suitable alternate cell line. We then turned our attention
9
back to the LNCaP cells. After numerous attempts using multiple cell culture
media/conditions (with and without sex hormone supplementation) , we identified
circumstances (RPMI with 10% FCS, complemented with non-essential amino acids,
sodium pyruvate, mercaptoethanol, and glutamine) which resulted in good cell growth and
cells that were readily 51Cr labeled and, importantly, retained the radiolabel. We are now
back on track with our immunization experiments and CTL evaluation. Because of these
delays, we have requested a no cost extension of the program in order to complete all these
originally proposed immunological evaluations.
Task 3. Prepare a PSMA covalent conjugate having a superior lipopeptide carrier
determined from Task 2. Perform mouse immunization experiments.
Test responses against prostate cancer cell-lines (months 20-36)
In view of our earlier difficulties with the PSA protein conjugates, we have replaced the
PSMA protein-conjugate work with relevant PSMA peptide epitope experiments. These
conjugates were synthesized (Figure 1) and their evaluation has been incorporated into
Task 2 which is underway.
C. Key Research Accomplishments
This section provides a list of key accomplishments to date of this research.
" Functionalized ligands recognized by TLR-2 and TLR-7 have been prepared.
These ligands were chemically modified with thiol-reactive haloacetamide
functionality for attachment of cysteine-containing antigenic proteins and
peptides.
"* TLR-7 and TLR-2 ligand-antigenic peptide conjugates have been prepared as
well as a TLR-7-ligand PSA protein conjugate.
"* Mouse immunization experiments are in progress. Data indicate an
immunogenic response to the antigenic epitopes.
D. Reportable Outcomes
This program supports graduate research assistant, Mr. Jiang Sha, and the results from his
research will be incorporated into his dissertation.
E. Conclusions
Research on this program thus far has provided modified TLR-2 and TLR-7 ligands suitable
for chemical attachment of prostate cancer associated antigenic peptides and proteins. The
resulting immunostimulating conjugates are being evaluated in mouse immunization
experiments in order to ascertain the production of CD8+ and CD4+ T-cells as well as
antibodies specific to prostate cancer antigens.
10